RESUMO
Research involving biosafety level 3 pathogens such as West Nile virus (WNV) is often limited by the limited space and technical constraints of these environments. To conduct complex analytical studies outside of high containment, robust and reliable inactivation methods are needed that maintain compatibility with downstream assays. Here we report the inactivation of WNV in spiked serum samples using a commercially available SDS-PAGE sample buffer for proteomic studies. Using this method, we demonstrate its utility by identification proteins differentially expressed in the serum of mice experimentally infected with WNV.
Assuntos
Proteínas Sanguíneas/metabolismo , Detergentes/farmacologia , Temperatura Alta , Proteômica/métodos , Substâncias Redutoras/farmacologia , Soro/virologia , Inativação de Vírus , Vírus do Nilo Ocidental/fisiologia , Animais , Soluções Tampão , Ditiotreitol/farmacologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Camundongos , Dodecilsulfato de Sódio/farmacologia , Tensoativos/farmacologia , Ensaio de Placa Viral , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/efeitos dos fármacosRESUMO
Identification of chromosomal markers for rapid detection of Bacillus anthracis is difficult because significant chromosomal homology exists among B. anthracis, Bacillus cereus, and Bacillus thuringiensis. We evaluated the bacterial gyrA gene as a potential chromosomal marker for B. anthracis. A real-time PCR assay was developed for the detection of B. anthracis. After analysis of the unique nucleotide sequence of the B. anthracis gyrA gene, a fluorescent 3' minor groove binding probe was tested with 171 organisms from 29 genera of bacteria, including 102 Bacillus strains. The assay was found to be specific for all 43 strains of B. anthracis tested. In addition, a test panel of 105 samples was analyzed to evaluate the potential diagnostic capability of the assay. The assay showed 100% specificity, demonstrating the usefulness of the gyrA gene as a specific chromosomal marker for B. anthracis.
Assuntos
Bacillus anthracis/genética , DNA Girase/genética , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Marcadores Genéticos , Dados de Sequência Molecular , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a method for identifying Bacillus anthracis by analyzing two chromosomal targets, the 16S-23S intergenic spacer region (ISR) and the gyrA gene. The 16S-23S ISR was analyzed by this method with 42 strains of B. anthracis, 36 strains of Bacillus cereus, and 12 strains of Bacillus thuringiensis; the gyrA gene was analyzed by this method with 33 strains of B. anthracis, 27 strains of B. cereus, and 9 strains of B. thuringiensis. Two blind panels of 45 samples each were analyzed to evaluate the potential diagnostic capability of this method. Our results show that DHPLC is an efficient method for the identification of B. anthracis.